Alteration of gene expression and DNA methylation in drug-resistant gastric cancer

  • Authors:
    • Osamu Maeda
    • Takafumi Ando
    • Naoki Ohmiya
    • Kazuhiro Ishiguro
    • Osamu Watanabe
    • Ryoji Miyahara
    • Yoko Hibi
    • Taku Nagai
    • Kiyofumi Yamada
    • Hidemi Goto
  • View Affiliations

  • Published online on: February 6, 2014     https://doi.org/10.3892/or.2014.3014
  • Pages: 1883-1890
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Abstract

The mechanisms of drug resistance in cancer are not fully elucidated. To study the drug resistance of gastric cancer, we analyzed gene expression and DNA methylation profiles of 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant gastric cancer cells and biopsy specimens. Drug-resistant gastric cancer cells were established with culture for >10 months in a medium containing 5-FU or CDDP. Endoscopic biopsy specimens were obtained from gastric cancer patients who underwent chemotherapy with oral fluoropyrimidine S-1 and CDDP. Gene expression and DNA methylation analyses were performed using microarray, and validated using real-time PCR and pyrosequencing, respectively. Out of 17,933 genes, 541 genes commonly increased and 569 genes decreased in both 5-FU- and CDDP-resistant AGS cells. Genes with expression changed by drugs were related to GO term ‘extracellular region’ and ‘p53 signaling pathway’ in both 5-FU- and CDDP-treated cells. Expression of 15 genes including KLK13 increased and 12 genes including ETV7 decreased, in both drug-resistant cells and biopsy specimens of two patients after chemotherapy. Out of 10,365 genes evaluated with both expression microarray and methylation microarray, 74 genes were hypermethylated and downregulated, or hypomethylated and upregulated in either 5-FU-resistant or CDDP-resistant cells. Of these genes, expression of 21 genes including FSCN1, CPT1C and NOTCH3, increased from treatment with a demethylating agent. There are alterations of gene expression and DNA methylation in drug-resistant gastric cancer; they may be related to mechanisms of drug resistance and may be useful as biomarkers of gastric cancer drug sensitivity.

Introduction

Gastric cancer is one of the most common causes of cancer-related mortality, responsible for >700,000 deaths worldwide per year (1). Although the main treatment strategy for gastric cancer is surgical or endoscopic resection, unresectable cases are treated with systemic chemotherapy. Platinum agents and fluoropyrimidine are the key therapeutic drugs for advanced gastric cancer (2) and drug resistance is an important problem accompanying treatment. A number of studies have previously reported on the mechanisms of gastric cancer chemoresistance (3) using cultured cells, animal models and clinical tissue samples. However, the mechanisms of drug resistance have not been fully elucidated.

It has been reported that both genetic and epigenetic changes play important roles in carcinogenesis and tumor progression (4). In various types of cancer, epigenetic changes are known to be early events in the multi-steps of carcinogenesis (5). Promoter hypermethylation is well-known to be important for the suppression of tumor suppressor gene expression (4). Mechanisms of cancer drug resistance are considered to be multifactorial; they have epigenetic alterations (6) and involve multiple gene functions and signaling pathways. A better understanding of such mechanisms may provide therapeutic strategies for gastric cancer.

In the present study, drug-resistant gastric cancer cell lines were established, and biopsy specimens were obtained from patients after the acquisition of drug resistance. Genome-wide analysis of gene expression and DNA methylation with a microarray for drug-resistant cell lines and endoscopic biopsy specimens of gastric cancer was performed. Validation with quantitative methods was also performed.

Materials and methods

Cell culture and 5-aza-2′-deoxycytidine (5-aza-dC) treatment

AGS was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and cultured in RPMI-1640 medium with 10% FBS at 37°C with 5% CO2. For treatment with 5-aza-dC (decitabine), cells were seeded on day 0, and exposed to freshly prepared 10 μmol/l 5-aza-dC (Sigma-Aldrich, Tokyo, Japan) for 24 h on days 1 and 3. After each treatment, the cells were placed in fresh medium and harvested on day 4 (7).

Drug-resistant gastric cancer cells

Resistant AGS cells were generated by continuous exposure to increasing concentrations of cisplatin (CDDP) or 5-fluorouracil (5-FU) for 10 months. Viability of cells was measured by MTS-formazan reduction using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). AGS cells (2×103) were cultured using 96-well microplates for 24 h, and exposed to various concentrations of CDDP or 5-FU for 48 h to calculate the IC50 of CDDP or 5-FU for each cell line.

Patients and biopsy specimens

Endoscopic biopsy specimens were obtained from two patients with unresectable advanced gastric cancer who underwent 3 and 4 courses of chemotherapy with oral fluoropyrimidine S-1 plus CDDP (8) before and after treatment. Samples after chemotherapy were obtained from lesions with viable cancer. The present study was approved by the Ethics Committee of Nagoya University Graduate School of Medicine, and written informed consent was provided by the patients.

Extraction of DNA and RNA from cell lines and gastric biopsy specimens

Cells or biopsy specimens were stored at −80°C for DNA extraction, and we used RNAlater (Ambion, Austin, TX, USA) for RNA extraction. For extraction of DNA and RNA, DNA Mini kit (Qiagen, Venlo, The Netherlands) and RNA Mini kit (Qiagen) were used, respectively.

Gene expression analysis with microarray

Expression analysis was performed with SurePrint G3 Human GE 8×60K (Agilent, Loveland, CO, USA). Expression of mRNA of autosomal 17,933 genes was evaluated with 23,856 corresponding probes. A difference in signal intensity >2-fold was judged to be significant.

DNA methylation microarray

Bisulfite-converted DNA was used for hybridization on Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA). The β-value [intensity of the methylated allele (M)/(intensity of the unmethylated allele (U) + intensity of the methylated allele (M) + 100)] was calculated for each CpG site (9). Methylation levels of candidate promoter lesions in CpG islands of 11,692 genes were evaluated. Genes with a difference in their β-value >0.1 were extracted. Of these 11,692 genes, expression of 10,365 genes was also evaluated with expression microarray.

Gene ontology (GO) and pathway analysis

GO analysis was performed with TargetMine (http://targetmine.nibio.go.jp/targetmine/begin.do; National Institute of Biomedical Innovation, Osaka, Japan), and pathway analysis was performed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GO terms or pathways with P<0.05 using the Holm-Bonferroni method were judged to be significantly enriched.

Real-time PCR

Real-time PCR was performed to validate expression of mRNA with TaqMan Gene Expression Assays and TaqMan Gene Expression Master Mix (both from Applied Biosystems, Foster City, CA, USA).

Bisulfite pyrosequencing

Bisulfite treatment was performed with the EpiTect kit (Qiagen) according to the manufacturer’s protocol. PyroMark PCR (Qiagen) was used to perform PCR, and bisulfite pyrosequencing was performed as previously reported (1012). In brief, the biotinylated PCR product was captured on streptavidin-coated beads (Amersham Biosciences, Uppsala, Sweden) and run on the PSQHS Pyrosequencing System (Biotage, Uppsala, Sweden) to obtain the degree of methylation.

Results

Resistance to CDDP or 5-FU of established drug-resistant cell lines

To confirm whether gastric cancer cells cultured in chemotherapy agents obtained drug resistance, IC50 values were measured. IC50 values of 5-FU were 10 and 56 μM in parent AGS and 5-FU-resistant AGS (5FUr), respectively. IC50 values of CDDP were 13 and 25 μM in parent AGS and CDDP-resistant AGS (CDDPr), respectively.

Genes with altered expression in drug-resistant cells and biopsy specimens

To characterize gene expression profiles of drug-resistant gastric cancer cells, expression microarray analysis was performed. A comparison of parent AGS, 5FUr and CDDPr change of expression is shown in Fig. 1. The expression of 541 genes increased and the expression of 569 genes decreased in both 5FUr and CDDPr compared with parent AGS. In contrast, the expression of only 25 genes increased in 5FUr and decreased in CDDPr, and the expression of only seven genes decreased in 5FUr and increased in CDDPr. To examine the characterization of genes with expression altered by drug treatment, we performed GO analysis and pathway analysis (Table I). Although most enriched GO terms differed between those changed in 5FUr and those changed in CDDPr, ‘extracellular region’ in GO terms of cellular component was commonly enriched in both 5FUr and CDDPr. With pathway analysis, the ‘p53 signaling pathway’ was enriched in both 5FUr and CDDPr.

Table I

Significantly enriched gene ontology (GO) terms and pathways of genes in which expression was changed in 5-fluorouracil-resistant AGS cells (5FUr) and cisplatin-resistant AGS cells (CDDPr), compared with parent AGS cells.

Table I

Significantly enriched gene ontology (GO) terms and pathways of genes in which expression was changed in 5-fluorouracil-resistant AGS cells (5FUr) and cisplatin-resistant AGS cells (CDDPr), compared with parent AGS cells.

A, GO terms (biological processes)

GO terms (biological processes)P-valueNo. of genes
Increased in 5FUr(No enrichment)
Increased in CDDPrResponse to other organism(GO:0051707)0.00078045
Cell surface receptor signaling pathway(GO:0007166)0.00125163
Response to virus(GO:0009615)0.0018031
Response to biotic stimulus(GO:0009607)0.0028845
Signal transduction(GO:0007165)0.00382247
Regulation of signal transduction(GO:0009966)0.00511125
Positive regulation of cell communication(GO:0010647)0.0070869
Positive regulation of signaling(GO:0023056)0.0070869
Immune system process(GO:0002376)0.00867130
Regulation of cell motility(GO:2000145)0.010541
Positive regulation of signal transduction(GO:0009967)0.012367
Regulation of cell migration(GO:0030334)0.014639
Single-organism process(GO:0044699)0.0151516
Regulation of signaling(GO:0023051)0.0159132
Regulation of cell communication(GO:0010646)0.0182132
Response to stimulus(GO:0050896)0.0206341
Regulation of response to stimulus(GO:0048583)0.0212157
Positive regulation of cell migration(GO:0030335)0.022427
Regulation of cellular component movement(GO:0051270)0.024443
Positive regulation of cell motility(GO:2000147)0.030827
Regulation of localization(GO:0032879)0.032492
Regulation of locomotion(GO:0040012)0.049641
Decreased in 5FUrDefense response(GO:0006952)2.86E-05110
Immune system process(GO:0002376)0.000327148
Innate immune response(GO:0045087)0.00036480
Single-multicellular organism process(GO:0044707)0.000918290
Immune response(GO:0006955)0.0131100
Multicellular organismal process(GO:0032501)0.0161295
Decreased in CDDPr Single-multicellular organism process(GO:0044707)0.0343279
Xenobiotic catabolic process(GO:0042178)0.04785

B, GO terms (cellular components)

GO terms (cellular components)P-valueNo. of genes

Increased in 5FUrExtracellular region(GO:0005576)4.64E-07130
Cell periphery(GO:0071944)0.000754280
Plasma membrane(GO:0005886)0.000907277
Extracellular region part(GO:0044421)0.0064770
Plasma membrane part(GO:0044459)0.0134136
Extracellular space(GO:0005615)0.027156
Increased in CDDPrExtracellular region(GO:0005576)0.037492
Decreased in 5FUrExtracellular region(GO:0005576)3.24E-05116
Decreased in CDDPrExtracellular region(GO:0005576)7.37E-08121
Cornified envelope(GO:0001533)0.01248
Intrinsic to membrane(GO:0031224)0.0175142
Integral to membrane(GO:0016021)0.0195138
Cell periphery(GO:0071944)0.0202243
Plasma membrane(GO:0005886)0.0268240

C, GO terms (molecular functions)

GO terms (molecular functions)P-valueNo. of genes

Increased in 5FUrSerine-type peptidase activity(GO:0008236)3.29E-0524
Serine hydrolase activity(GO:0017171)5.16E-0524
Serine-type endopeptidase activity(GO:0004252)0.0032517
Increased in CDDPr(No enrichment)
Decreased in 5FUr(No enrichment)
Decreased in CDDPrReceptor binding(GO:0005102)5.08E-0483
Cytokine activity(GO:0005125)0.038315

D, pathway

PathwayP-valueNo. of genes

Increased in 5FUrp53 signaling pathway0.022214
Increased in CDDPrp53 signaling pathway8.56E-0718
Decreased in 5FUr(No enrichment)
Decreased in CDDPrCytokine-cytokine receptor interaction0.0061837

To compare gene expression of gastric cancer before and after chemotherapy, expression microarray analysis with endoscopic biopsy specimens was performed. Genes with altered expression both in drug-resistant cells and in biopsy specimens after chemotherapy were extracted. The expression of 15 genes increased 5FUr, CDDPr and two pairs of biopsy specimens, and the expression of 12 genes decreased (Fig. 2, Table II).

Table II

List of genes in which expression was changed commonly in drug-resistant cells and biopsy specimens.

Table II

List of genes in which expression was changed commonly in drug-resistant cells and biopsy specimens.

Increased in 5FUr and two biopsy specimens after chemotherapyDecreased in 5FUr and two biopsy specimens after chemotherapyIncreased in CDDPr and two biopsy specimens after chemotherapyDecreased in CDDPr and two biopsy specimens after chemotherapyIncreased in 5FUr, CDDPr, and two biopsy specimens after chemotherapyDecreased in 5FUr, CDDPr, and two biopsy specimens after chemotherapy
APOC1ALPK1ACTG2ACOXLAPOC1ALPK1
BAIAP3C17orf110ANPEPALPK1CRYMCCL21
C4BPAC4orf47APOC1BATFDNAJC28CYP2E1
C6orf154CCL21C9orf123BEST4HSD17B6ETV7
CAPS2CYP2E1CELA3BCCL21IQCDFBXO15
CFTRETV7CRYMCYP2E1KLK13GPR110
CRYMFBXO15CTSGETV7KREMEN2NLRC5
DNAJC28GPR110DNAJC28FBXO15OLFML3PLIN4
FRZBHEPACAM2HOXB3GPR110OTUD7ASLC22A20
HSD17B6 IFI44LHSD17B6INSCPHACTR3SLC26A9
IGF1KRT6CIQCDKRT31PLATSLC28A3
IP6K3LAMC2KLK13MUC1RARRES2SNORA22
IQCDNLRC5KREMEN2NCKAP5SRI
KCTD7OR52K2NLRP2NLRC5TAC3
KLK13PLIN4NRG1PCSK9TNNI3
KREMEN2SLC22A20OLFML3PLIN4
LAMA1SLC26A9OTUD7ARAB27B
LRRC6SLC28A3PHACTR3SLC22A20
MSLNSNORA22PLATSLC26A9
NR2F1SPRR3RARRES2SLC28A3
OLFML3ZNF750RASL10ASLFNL1
OOEPSRISNORA22
OTUD7ATAC3SYT13
PHACTR3TNFSF9
PLATTNNI3
PRODH
RARRES2
SCN2A
SEPP1
SRI
TAC3
TNNI3

[i] 5FUr, 5-fluorouracil-resistant AGS cells; CDDPr, cisplatin-resistant AGS cells.

To validate the gene expression change extracted with microarray, real-time PCR for KLK13 and ETV7 was performed. Consistent with microarray analysis, KLK13 increased in both drug-resistant AGS cells and endoscopic biopsy specimens after chemotherapy (Fig. 3A). In contrast, ETV7 decreased in both drug-resistant cells and biopsy specimens after treatment (Fig. 3B).

Integrated analysis of expression and methylation microarray

To study whether DNA methylation contributes to gene expression change caused by chemotherapy agents, we analyzed the methylation profiles of 5FUr and CDDPr and compared them with parent AGS. The number of genes that were hypermethylated and decreased in expression and that were hypomethylated and increased in expression was 74.

Next, to evaluate whether alterations in the DNA methylation of these genes was related to expression change, gene expression change was measured by treatment with a demethylating agent. Twenty-one of those 74 genes increased in expression after treatment with decitabine (Table III). Furthermore, gene expression and methylation were validated with quantitative methods, TaqMan PCR and bisulfite pyrosequencing, respectively, for FSCN1, CPT1C and NOTCH3. Expression of these three genes increased after treatment with decitabine (Fig. 4). FSCN1 revealed increased expression and hypomethylation in CDDPr compared with parent AGS cells. Regarding CPT1C, 5FUr showed hypomethylation and increased expression. CDDPr also showed increased expression, although the methylation level did not change. NOTCH3 showed increased expression and hypomethylation, especially in 5FUr.

Table III

List of genes in which expression increased and methylation levels decreased, or expression decreased and methylation levels increased.

Table III

List of genes in which expression increased and methylation levels decreased, or expression decreased and methylation levels increased.

Increased expression and hypo- methylation in 5FUrDecreased expression and hyper- methylation in 5FUrIncreased expression and hypo- methylation in CDDPrDecreased expression and hyper- methylation in CDDPr
ARCATP2C2ABCG4ATP2C2
C12orf34C15orf60C12orf34C15orf60
CPT1CPRAMECARD9FRMD6
CST6ZNF773CST6SECTM1
CYB5R2FESTNFSF12
KCNH8FSCN1ZNF773
MESP1KCNH8
NOTCH3MESP1
RASGRP2VGF
TNFSF12

[i] 5FUr, 5-fluorouracil-resistant AGS cells; CDDPr, cisplatin-resistant AGS cells.

Discussion

The gene expression and the DNA methylation of drug-resistant cell lines and biopsy specimens were evaluated before and after chemotherapy, and some genes revealed altered expression and altered methylation. In drug-resistant cells, treatment with 5-FU and CDDP caused consistent expression change in >1,000 genes. In contrast, the expression of only a small number of genes changed reciprocally (Fig. 1), and those genes were considered to be potentially related with drug-specific sensitivity. In GO analysis, only a small number of GO terms are commonly enriched in both 5-FU-resistant cells and CDDP-resistant cells, and enriched pathways related to the two drugs are also different. These findings may be related to differences in mechanisms of resistance to each drug.

Expression change of genes before and after chemotherapy was also evaluated using endoscopic biopsy specimens, and revealed that profiles of changes were different in the two patients. Expression of some genes increased or decreased, both in drug-resistant cells and biopsy specimens after chemotherapy (Fig. 2, Table II). Such genes are considered to be candidates as key molecules for drug resistance, and may be useful as biomarkers of drug-sensitivity.

KLK13 is one member of the tissue kallikrein (KLK) family which includes 15 genes (KLK1-KLK15) and plays a role in tumor cell invasion and migration (13). KLK13 has already been reported to be upregulated in gastric cancer cells after exposure to antineoplastic agents, including epirubicin and methotrexate (14). It has also been reported that overexpression of KLK13 results in an increase of malignant cell behavior, and that knockdown of its endogenous gene expression causes a significant decrease in cell migratory and invasive properties (13). We found that KLK13 increased in both drug-resistant cells and biopsy specimens, a finding suggesting that KLK13 may play a role in 5-FU and CDDP resistance in gastric cancer.

In contrast to KLK13, expression of ETV7 decreased in drug-resistant cells. ETV7 is a member of the Ets transcription factor family and is reported to act as an inhibitor of differentiation (15). Since ETV7 was downregulated in drug-resistant gastric cancer in the present study, it may be related with mechanisms of gastric cancer drug-sensitivity.

It has been reported that epigenetic profiles are useful for identifying molecular mediators for cancer drug sensitivity (6). In terms of a correlation between gene expression and DNA methylation, we also performed expression and methylation microarray analyses, and found some genes with altered methylation levels and expression levels. FSCN1 revealed increased expression and hypomethylation in CDDP-resistant cells. FSCN1 has been reported to play an important role in cancer development and is associated with invasion and metastasis (16). It has also been reported that higher intensity FSCN1 staining correlated with more-advanced cancer stages, and inversely correlated with survival rates in gastric adenocarcinoma (17). This suggests that FSCN1 may influence patient survival through acquisition of resistance to chemotherapy drugs.

In our experiment, CPT1C was increased in expression in 5-FU and CDDP-resistant cells, and demethylated in 5-FU-resistant cells. CPT1C has been reported to promote tumor growth and rapamycin resistance (18). CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently upregulated in human lung tumors (18). To our knowledge, there has been no report of a relationship between CPT1C and 5-FU or CDDP.

Notch is a transmembrane heterodimeric receptor with 4 distinct members (NOTCH1 to NOTCH4) present in humans. NOTCH3 is one of the Notch family members. It has been reported that NOTCH1, another molecule in Notch family members, expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro, and also with poor survival in human esophageal squamous cell carcinomas (19). It has also been reported that expression levels of Notch3 were increased in rat tracheal epithelial cells after treatment with 5-FU (20). NOTCH3 knockdown enhanced the sensitivity of nasopharyngeal carcinoma cells to CDDP treatment (21), and NOTCH3 overexpression correlated with shorter progression-free/overall survival in patients with advanced stage ovarian carcinoma treated with platinum and taxane (22). In our data, NOTCH3 was upregulated in drug-resistant cells. In gastric cancer, NOTCH3 may be related to drug resistance.

However, we could not find a significant difference in methylation levels among biopsy samples. One of the limitations is that biopsy specimens may not represent the characteristics of the whole tumor, since gastric cancer is known to be biologically heterogeneous.

In the present study, genes with altered expression and DNA methylation were extracted after treatment with chemotherapeutic agents in gastric cancer. These alterations may be related to mechanisms of gastric cancer drug resistance, and may be useful as biomarkers that predict drug sensitivity. Further studies with a large number of clinical samples are necessary.

Acknowledgements

The authors thank Ms. Chie Moriyama for her technical support. This study was funded by the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 20378053).

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2014-April
Volume 31 Issue 4

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Spandidos Publications style
Maeda O, Ando T, Ohmiya N, Ishiguro K, Watanabe O, Miyahara R, Hibi Y, Nagai T, Yamada K, Goto H, Goto H, et al: Alteration of gene expression and DNA methylation in drug-resistant gastric cancer. Oncol Rep 31: 1883-1890, 2014
APA
Maeda, O., Ando, T., Ohmiya, N., Ishiguro, K., Watanabe, O., Miyahara, R. ... Goto, H. (2014). Alteration of gene expression and DNA methylation in drug-resistant gastric cancer. Oncology Reports, 31, 1883-1890. https://doi.org/10.3892/or.2014.3014
MLA
Maeda, O., Ando, T., Ohmiya, N., Ishiguro, K., Watanabe, O., Miyahara, R., Hibi, Y., Nagai, T., Yamada, K., Goto, H."Alteration of gene expression and DNA methylation in drug-resistant gastric cancer". Oncology Reports 31.4 (2014): 1883-1890.
Chicago
Maeda, O., Ando, T., Ohmiya, N., Ishiguro, K., Watanabe, O., Miyahara, R., Hibi, Y., Nagai, T., Yamada, K., Goto, H."Alteration of gene expression and DNA methylation in drug-resistant gastric cancer". Oncology Reports 31, no. 4 (2014): 1883-1890. https://doi.org/10.3892/or.2014.3014